Regulation und Transport der Disintegrin und Metalloproteinasen ADAM10 und ADAM17

• Regulation and transport of the disintegrin and metalloproteinases ADAM10 and ADAM17

Groth, Esther; Ludwig, Andreas (Thesis advisor); Fabry, Marlies (Thesis advisor); Pradel, Gabriele (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen, 2016

Abstract

By mediating proteolytic shedding of several transmembrane proteins like growth factors, cytokines, adhesion molecules, and receptors on cell surface, the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of different diseases like Alzheimer, asthma, cardiovascular disease, cancer, and inflammation. Furthermore, they play a pivotal role in the regulation of embryogenesis and the development of an organism. Their own regulation is subject to several mechanisms like gene induction, conformational changes, intracellular maturation and transport of the proteases, changes of subcellular localization, or interaction with adapter proteins. ADAM10 and ADAM17 are the best investigated proteases of the ADAM-family but their regulation is still not fully understood. In particular, less is known how ADAM10 and ADAM17 can be regulated on the surface expression level and which mechanisms could be implicated.This PhD thesis investigates the stimulus-dependent surface expression of ADAM10 and ADAM17 in the human alveolar tumor epithelial cell line A549. The surface regulation of ADAM10 was not affected by cell stimulation with IFNγ/TNFα, LPS, bleomycin, and PMA, whereas ADAM17 surface expression was upregulated by bleomycin and IFNγ/TNFα cell treatment. In contrast, stimulation with PMA led to the downregulation of maturated ADAM17 on the surface. In parallel, the pro form of ADAM17 was increased in the cell lysates which was in line with an increased gene expression of ADAM17. Despite a surface reduction of mature ADAM17, the proteolytic activity of the protease was enhanced, as measured by syndecan-1 and -4 shedding. The PMA effect on ADAM17 surface regulation was slightly suppressed by inhibition of metalloproteinases or ERK1/2, but a major proportion of the PMA effect still remained in the presence of the inhibitors. It could be demonstrated for the first time that the stimulation with PMA is not only accompanied by a sustained reduction of ADAM17 on cell surface but also by a release of the mature ADAM17 form in exosomes. The released vesicles contained not only the mature ADAM17 but also the exosomal markers CD9, CD63, hsp70, and flotillin-1 and the vesicles were characterized by a density of 1.12 to 1.14 g/ml in the sucrose gradient, which indicates that they were exosomes. Furthermore, α5-integrin was detectable in the exosomes after stimulation. The exosomal ADAM17 release was also observed in monocytic THP-1 cells stimulated with LPS, indicating that such release may occur under pathophysiologic conditions in several cell types. ADAM10 was released constitutively in all cases. Not only the mature form but also the pro form was detectable. The biogenesis of ADAM17-containing exosomes seems to be dynamin-independent, as shown by inhibition experiments and knockdown of dynamin via shRNA and siRNA. But ADAM17 is co-localized with flotillin-1 in lipid rafts and in the PMA-induced exosomes. Therefore, an internalization process which presumably precedes the exosome formation and release of ADAM17 could be flotillin-1-associated. The investigations of the cytoplasmic domain of ADAM17 revealed that the deficiency of the phosphorylation sites threonine 735, serine 794, and serine 822 has no effect on PMA-induced JAM-A shedding or on PMA-induced exosomal release of ADAM17. But the complete deletion of the ADAM17 C-terminus could prevent the release of the protease in exosomes. Flow cytometry analysis of PMA-induced exosomes indicated that at least a part of the ADAM10 and ADAM17 molecules are orientated on the exosomal surface with their protease domain outside. Despite this orientation, the analysis of syndecan shedding with exosomes of knockout cells demonstrated that exosomal ADAM17 does not participate in the shedding of syndecans while syndecan shedding by ADAM10 could be observed.A new regulation mechanism for ADAM17 was revealed. Its physiological function has to be examined in further investigations.